CN109793750B - Anti-tumor nano-drug - Google Patents
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- CN109793750B CN109793750B CN201910246814.6A CN201910246814A CN109793750B CN 109793750 B CN109793750 B CN 109793750B CN 201910246814 A CN201910246814 A CN 201910246814A CN 109793750 B CN109793750 B CN 109793750B
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Abstract
The invention provides an anti-tumor compound, which is composed of a DNA tetrahedral structure and HApt according to the molar ratio of 1: 1; the HApt is a single-chain nucleic acid molecule which can target, recognize and bind to HER2 protein and mediate HER2 protein to enter a cell lysosome for degradation; the HApt is linked to 1 of 4 single strands constituting the DNA tetrahedral structure by a covalent bond. The anti-tumor compound can be specifically taken by HER2 positive tumor cells, can effectively inhibit the proliferation of the tumor cells and promote the apoptosis of the tumor cells, and has good application prospect in the preparation of anti-tumor drugs.
Description
Technical Field
The invention relates to the field of antitumor drugs, and particularly relates to a HER2 targeted antitumor drug.
Background
The Epidermal growth factor receptor (EGFR, HER) family has 4 members (HER1, HER2, HER3, HER4), and these receptors are mainly present on cell membranes and are closely related to cell proliferation, apoptosis, and the like. Among them, the HER2 protein is frequently overexpressed in breast cancer, gastric cancer, head and neck tumors, etc., and most frequently expressed in malignant breast cancer, whereas HER2 is not expressed or is underexpressed in normal tissues. Overexpression of HER2 causes abnormal cell proliferation and differentiation, and further causes the conditions of faster disease course and poorer prognosis of breast cancer patients. Therefore, the determination of the effects of overexpression and inhibition of HER2 as early as possible has great significance for diagnosis and treatment of HER2 positive malignant breast cancer patients.
The current therapeutic drugs for HER2 positive malignant breast cancer are mainly antibodies, such as trastuzumab and the like; however, such antibody drugs cause many adverse reactions such as diarrhea, hepatotoxicity, etc. In addition, the production cost of such antibody drugs is high, the procedures are complicated, and the preservation manner of such antibodies is also complicated. Therefore, the development of new drugs for treating HER2 positive breast cancer is of great importance.
At present, the DNA aptamer single strand (anti-HER2 aptamer, HApt) of HER2 protein screened by SELEX technology can target HER2 protein and has a certain anti-tumor effect. The HApt can target and recognize and bind to HER2 protein, and mediate HER2 protein to enter a cell lysosome to be degraded, so that the amount of HER2 protein of a cell is reduced, the proliferation of the cell is inhibited, and the apoptosis is promoted. However, single-stranded HApt still has some disadvantages, such as poor structural stability, low cell-entry efficiency, and short action time.
Disclosure of Invention
In order to solve the above problems, the present invention provides an anti-swelling complex consisting of a DNA tetrahedral structure and HApt in a molar ratio of 1: 1;
the HApt is a single-chain nucleic acid molecule which can target, recognize and bind to HER2 protein and mediate HER2 protein to enter a cell lysosome for degradation;
the HApt is connected to 1 single strand of 4 single strands forming a DNA tetrahedral structure through a covalent bond;
the 4 single-stranded sequences are different from each other.
The anti-tumor complex as described above, wherein the sequence of the HApt is more than 90% homologous to the sequence of SEQ ID NO. 5.
The anti-tumor complex as described above, wherein the sequence of the HApt is the sequence shown in SEQ ID NO. 5.
The anti-tumor compound as described above, wherein the sequences of the 4 single strands are selected from the sequences of SEQ ID NO. 1-4 one to one.
The anti-tumor complex as described above, wherein the HApt and the single-chain linked thereto further comprise a linker sequence.
The anti-tumor complex as described above, wherein a fluorescent molecule Cy5 is further linked to the 5' end of each single strand.
The invention also provides a preparation method of the compound, which is characterized in that 4 single strands of the DNA tetrahedron are placed at a temperature which is enough for denaturation to be maintained for more than 10min, and then the temperature is reduced to 2-8 ℃ and maintained for more than 20 min;
1 of the 4 single strands is linked to a HApt sequence.
The invention also provides a preparation method of the compound, which is to place 4 single strands of the DNA tetrahedron at 95 ℃ for 10-15 min, and then reduce the temperature to 4 ℃ for 20 min.
The invention also provides application of the anti-tumor compound in preparing anti-tumor drugs.
In the application, the anti-tumor drug is a drug for resisting HER2 positive tumor.
The invention also provides an anti-tumor medicament which is prepared by taking the anti-tumor compound as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
The anti-tumor medicament is a medicament for resisting HER2 positive tumors.
The invention has the following beneficial effects:
1) the complexes of the invention are specifically taken up by HER2 positive cells.
2) The compound of the invention can obviously inhibit the growth of HER2 positive tumor cells and mediate the apoptosis of the HER2 positive tumor cells.
3) The complexes of the invention do not cause apoptosis in HER2 negative cells.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The foregoing aspects of the present invention are explained in further detail below with reference to specific embodiments. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1: and (3) synthesis and preliminary identification of the HApt-tFNA nano robot. A, a synthetic schematic diagram of HApt-tFNA; b, an electrophoretogram of substances formed by pairing HApt-tFNA and related single chains; c, serum stability assay map of HApt-tFNA; d, visualization of the circulation in vivo of HApt-tFNA.
FIG. 2: HApt-tFNA and its related components make up a single-chain electropherogram.
FIG. 3: HApt-tFNA dynamic light scattering detection map.
FIG. 4: HApt-tFNA transmission electron microscopy images.
FIG. 5: HER2 test and tolerance test of different concentrations of HApt-tFNA (action 72h) of 3 cell lines.
FIG. 6: tolerance tests were carried out on 3 cell lines at different concentrations of HApt-tFNA (24, 48h effect).
FIG. 7: effect of 250nM tFNA on SK-BR-3 cell activity.
FIG. 8: graphs of uptake of Cy5-HApt and Cy5-HApt-tFNA by 3 cell lines.
FIG. 9: cy5-tFNA uptake assay in SK-BR-3 cells.
FIG. 10: cy5-tFNA uptake assay by MCF-7 cells.
FIG. 11: cy5-tFNA uptake assay by MCF-10A cells.
FIG. 12: cell cycle, apoptosis, and HER2 expression of SK-BR-3 cells were examined by Free HApt and HApt-tFNA. A-B, cell cycle detection; c, detecting cell apoptosis; D-F, HER2 expression profile.
FIG. 13: cell cycle and apoptosis of MCF-7 cells by Free HApt and HApt-tFNA were examined. A-B, cell cycle detection; and C, detecting apoptosis.
FIG. 14: cell cycle and apoptosis of MCF-10A cells by Free HApt and HApt-tFNA were examined. A-B, cell cycle detection; and C, detecting apoptosis.
FIG. 15: and HApt subcellular localization by Cy5-HApt and Cy 5-HApt-tFNA. A, Cy5-HApt and Cy5-HApt-tFNA positioning contrast map; b, mapping of Cy5-HApt-tFNA within 6-36 h; c, mapping of Cy5-HApt-tFNA to HER2 at Cy5-HApt- tFNA treatment 12 and 24 h.
FIG. 16: mapping of Cy5-tFNA in SK-BR-3 cells.
FIG. 17: one slice of the positioning comparison graph (lower column) corresponding to the processing 24h in fig. 15C.
FIG. 18: graphs were examined for the effect of HApt-fna on HER2 under the influence of chloroquinine.
FIG. 19: detection of PI3K/AKT signaling pathway-related proteins in SK-BR-3 cells by HApt-tFNA.
Detailed Description
Example Synthesis and characterization of HApt-tFNA
1. Synthesizing: four DNA single strands (S1, S2, S3, HApt-S4) were dissolved at the same final concentration in a TM buffer solution containing Tris-HCl and MgCl2 at concentrations of 10mM and 50mM, respectively, and the pH of the solution was adjusted to 8.0. And then, placing the mixture into a PCR instrument after vortex, uniform mixing and centrifugation, rapidly increasing the temperature to 95 ℃, stabilizing for 10-15 min, and then cooling to 4 ℃, and stabilizing for 20 min. I.e. a tetrahedral structure as shown in figure 1 is synthesized. In order to realize the fluorescence detection of tFNA or HApt-tFNA, a fluorescent group can be added to the 5' end of any single strand for synthesizing the tFNA or HApt-tFNA, the Cy5 fluorescent group is uniformly used in the place of the fluorescence detection, and the corresponding fluorescence labeling molecules are named Cy5-tFNA and Cy 5-HApt-tFNA.
TABLE 1 Single-stranded sequences
2. Identification
(1) Gel electrophoresis
a. Preparing polyacrylamide gel: mixing 40 wt% acrylamide solution, 10 XTAE, 10 wt% ammonium sulfate solution, distilled water and tetramethyl ethylene diamine according to the volume ratio of 1-2: 1 to prepare polyacrylamide gel.
b. Sample adding and electrophoresis: 1ul of 6 × loading buffer was mixed with 5ul of sample and marker, and then added to the corresponding electrophoresis tank. Electrophoresis was carried out for 80min under ice bath and constant pressure of 80V.
c. GelRed staining and exposure: and placing the polyacrylamide gel in a mixed liquid obtained by mixing GelRed and distilled water according to the ratio of 1: 50, keeping out of the sun, and shaking for 15-25 min. And (6) exposing.
As a result: FIG. 1A shows a schematic diagram of the synthesis of HApt-tFNA; as shown in FIG. 1B, lanes 1-S4, 2-HApt-S4, 3-S1, 4-S1+ S2, 5-S1+ S2+ S3, 6-S1+ S2+ S3+ S4(6-tFNA), 7-S1+ S2+ S3+ HApt-S4 (7-HApt-tFNA). Such as 1-S1, 2-S2, 3-S3, 4-S4, 5-HApt, 6-HApt-S4 shown in FIG. 2.
(2) Dynamic light scattering
Taking appropriate amount of tFNA and HApt-tFNA sample solutions, respectively, placing in a dynamic light scattering detector, and detecting.
As a result: as shown in FIG. 3, tFNA had a particle size of about 11nm, and HApt-tFNA had a particle size of about 20 nm.
(3) Transmission electron microscope
And (3) taking a proper amount of sample liquid on a metal sheet, irradiating for 5-10min under infrared to dry the sample liquid, and detecting on a machine.
As a result: as shown in FIG. 4, the shape of HApt-tFNA is approximately triangular under a transmission electron microscope, and the particle size is within 20 nm.
(4) Serum stability test
HApt-tFNA and free HApt were placed in 10% fetal calf serum, and after 0, 2, 6, 12, 24, and 36h at 37 deg.C, the remaining amount was checked by gel electrophoresis on PAGE gel.
As a result: as shown in FIG. 1C, HApt-tFNA has better serum stability than free HApt.
(5) In vivo circulation experiment
Cy5-HApt-tFNA and free Cy5-HApt were injected into 20g of nude mice (male, balb/c), 100ul (1 uM) per nude mouse, and the distribution and fluorescence intensity of the material in the nude mice were observed using a live mouse imager 2, 5, 10, 20, 30, 45, and 60min after injection.
As a result: as shown in FIG. 1D, HApt-tFNA was present in nude mice for a longer time than free HApt, and had more stable in vivo circulation stability.
The beneficial effects of HApt-tFNA will be further described in the form of experimental examples below.
The 3 cell lines involved in the experimental examples were: SK-BR-3: HER2 positive human breast cancer cells; MCF-7: HER2 negative human malignant breast cancer cells; MCF-10A: HER2 negative human normal breast cells.
Since Cy5 only serves as a fluorescent label, the effect of HApt-tFNA and Cy5-HApt-tFNA on cells in the experimental examples was considered to be equivalent.
Experimental example 1 identification of HER2 expression level and screening of HApt-tFNA optimum action concentration
Identification of the expression level of HER2
Immunofluorescence techniques:
A. the cell suspension (SK-BR-3, MCF-7, MCF-10A) is respectively inoculated in a confocal dish and placed in an incubator for 24 h. The medium was aspirated off, washed 3 times with PBS, 5min each;
fixing with 4% paraformaldehyde for 25min, removing paraformaldehyde by suction, washing with PBS for 5min for 3 times;
treating with 0.5% Triton-100 for 20-25min, removing Triton-100, washing with PBS for 3 times (5 min each);
D. treating sheep serum for 1h, removing sheep serum by suction, washing with PBS for 3 times, each for 5 min;
E. primary anti (anti-HER2 antibody) treatment was performed at 4 ℃ overnight. The next day, rewarming at 37 ℃ for 0.5h, recovering the primary antibody, washing with PBS 3 times, 5min each time. Treating the secondary antibody carrying fluorescence at 37 deg.C in dark for 1 hr, removing the secondary antibody, washing with PBS for 3 times, each for 5 min;
F. treating phalloidin in dark for 10-30min, removing phalloidin, washing with PBS for 5min for 3 times;
DAPI treatment, protected from light, 10min, aspiration of DAPI, washing 3 times with PBS, 5min each. Sealing with 10% glycerol, and storing at 4 deg.C in dark. And (6) performing detection on the machine.
As a result: as shown in FIG. 5A, HER2 protein was highly expressed in SK-BR-3 cells and was less expressed in MCF-7 and MCF-10A cells.
Screening for the optimal working concentration of HApt-tFNA
CCK-8 experiment
A. The cell suspension (SK-BR-3, MCF-7, MCF-10A, 100. mu.l/well) was inoculated into a 96-well plate, and the plate was pre-incubated in an incubator for 24 hours (37 ℃, 5% CO)2)。
B. The cultured cell suspension was divided into a blank control group, a positive control group (free HApt) and an experimental group (HApt-tFNA), and different concentrations (62.5, 125, 250, 375nM) were added to the experimental group, and an equal amount of PBS was added to the control group, followed by 24, 48, 72h (37 ℃, 5% CO) culture in an incubator2)。
C. Adding CCK-8 solution (10 mu l/well) into the experimental group and the control group respectively, and then incubating in an incubator for 1-4 h (37 ℃, 5% CO)2) And then the absorbance of each well was measured at 450 nm.
D. After the optimum concentration was selected, the effect of tFNA on the cell activity of SK-BR-3 cells was tested by the method described above.
As a result: as shown in FIG. 5B and FIG. 6, 250nM HApt-tFNA had significant inhibitory effect on SK-BR-3, but not on MCF-7 and MCF-10A; whereas 375nM HApt-tFNA had a significant inhibitory effect on MCF-10A, 250nM was chosen as the optimal effect concentration. As shown in FIG. 7, 250nM tFNA had no significant inhibitory effect on SK-BR-3.
Experimental example 2 uptake differences of three cells for Cy5-HApt-tFNA, free Cy5-HApt and Cy5-tFNA
1. Fluorescent tracing technique
A. Inoculating cell suspension (SK-BR-3, MCF-7, MCF-10A) into the confocal cuvette, and pre-culturing in incubator for 24h (37 deg.C, 5% CO)2)。
B. Cy5-HApt-tFNA, free Cy5-HApt and Cy5-tFNA were added at a concentration of 250nM and incubated in an incubator for 12 hours (37 ℃, 5% CO)2)。
C. Removing the culture medium by suction, washing with PBS for three times, 5min each time, fixing with 4 wt% paraformaldehyde for 25min, removing paraformaldehyde by suction, and washing with PBS for three times, 5min each time; treating with phalloidin, keeping out of the sun for 10-30min, removing phalloidin, and washing with PBS for 5min three times; then treating with DAPI, keeping out of the sun for 10min, removing DAPI by suction, and washing with PBS for 5min three times; sealing with 10 wt% glycerol, protecting from light, storing at 4 deg.C, and detecting on machine.
As a result: as shown in FIG. 8A, FIG. 9A, FIG. 10A, FIG. 11A, Cy5-HApt-tFNA and free Cy5-HApt can be ingested by SK-BR-3, but the uptake of Cy5-HApt-tFNA is significantly greater; MCF-7 and MCF-10A had uptake of both Cy5-HApt-tFNA and free Cy5-HApt, but significantly less; SK-BR-3, MCF-7 and MCF-10A all have more intake of tFNA.
2. Flow cytometry
A. Inoculating cell suspension (SK-BR-3, MCF-7 and MCF-10A) into 6-well plate, pre-culturing in incubator for 24 hr (37 deg.C, 5% CO)2)。
B. Cy5-HApt-tFNA, free Cy5-HApt and Cy5-tFNA were added at a concentration of 250nM and incubated in an incubator for 12 hours (37 ℃, 5% CO)2)。
C. The cells were collected, centrifuged at 1000r/min for 5min, resuspended in PBS, repeated three times, and tested on the machine.
As a result: as shown in FIG. 8B, FIG. 9B, FIG. 10B and FIG. 11B, both Cy5-HApt-tFNA and free Cy5-HApt were ingested by SK-BR-3, but the uptake of Cy5-HApt-tFNA was significantly greater; MCF-7 and MCF-10A had uptake of both Cy5-HApt-Tfna, free Cy5-HApt, but significantly less; SK-BR-3, MCF-7 and MCF-10A all have more intake of tFNA.
Notably, uptake of Cy5-HApt-tFNA was much lower for MCF-7 and MCF-10A, which were HER2 negative, than for tFNA. As a drug carrier, the tFNA drug-loaded complex should be readily accessible to the cells at least to the same extent as tFNA alone. However, it was unexpected that the complex of tFNA and HApt selectively "refused" to enter HER2 negative cells. The effect is more beneficial to the uptake of the medicine by HER2 positive cells due to the fact that the medicine is saved from randomly entering normal cells.
In addition, the uptake rate of Cy5-HApt-tFNA by HER2 positive SK-BR-3 is significantly higher than that of tFNA and HApt alone respectively, which indicates that HApt and tFNA promote each other and increase the drug uptake by HER2 positive cells.
In summary, HER2 negative cells gave small HApt-tFNA intake, and HER2 positive cells gave large HApt-tFNA intake.
Experimental example 3 Effect of comparative HApt-tFNA and free HApt on the cycle and apoptosis of three cells (SK-BR-3, MCF-7 and MCF-10A)
1. Flow cytometry-cell cycle
A. Inoculating cell suspension (SK-BR-3, MCF-7 and MCF-10A) into 6-well plate, pre-culturing in incubator for 24 hr (37 deg.C, 5% CO)2)。
B. Adding 250nM HApt-tFNA, free HApt, and culturing in incubator for 48h (37 deg.C, 5% CO)2)。
C. Collecting cells, centrifuging at 1000r/min for 5min, resuspending with PBS, repeating for three times, fixing the prepared single cell suspension with 70% ethanol, storing at 4 deg.C, and washing off the fixing solution with PBS before staining. Adding RNase A in 37 deg.C water bath for 30 min. Adding PI for dyeing, and mixing, and keeping away from light at 4 deg.C for 30 min. And (4) performing detection on the machine, and recording red fluorescence detection at the position of 488nm of the excitation wavelength.
As a result: as shown in FIGS. 12A-B, 13A-B and 14A-B, HApt-tFNA can significantly reduce the cell ratio in S phase of SK-BR-3 cells compared with free HApt, i.e., can more effectively inhibit cell cycle and proliferation; in MCF-7 and MCF-10A cells, HApt-tFNA can remarkably increase the cell proportion of MCF-7 and MCF-10A cells in S phase compared with free HApt, namely, the cell cycle and proliferation can be more effectively promoted.
2. Flow cytometry-apoptosis
A. Inoculating cell suspension (SK-BR-3, MCF-7 and MCF-10A) into 6-well plate, pre-culturing in incubator for 24 hr (37 deg.C, 5% CO)2)。
B. Adding 250nM HApt-tFNA, free HApt, and culturing in incubator for 48h (37 deg.C, 5% CO)2)。
C. Trypsinizing, collecting cells, centrifuging at 1000rpm for 5min, and removing the culture medium. Cells were washed twice with cold PBS, 1000rpm, 5min, and centrifuged twice. Cells were suspended in 400. mu.L of 1 × Annexin V binding solution at a concentration of approximately 1 × 106cells/mL. 50 μ L Annexin-FITC staining solution was added to the cell suspension, mixed gently, and incubated at 4 ℃ for 15min in the dark. Adding 5-10 μ L PI staining solution, mixing, and incubating at 4 deg.C in dark for 5 min. Immediate flow cytometryAnd (6) detecting by an instrument.
As a result: as shown in fig. 12C, 13C and 14C, HApt-tFNA can significantly increase the ratio of the SK-BR-3 cells to early apoptosis, late apoptosis and apoptosis compared to free HApt, i.e., can more effectively promote apoptosis; in MCF-7 and MCF-10A cells, the cells are not obviously apoptotic in early stage, apoptotic in late stage and the proportion of apoptotic cells is not obviously changed. That is, HApt-tFNA had no significant effect on apoptosis in both cells compared to free HApt.
Changes in HER2 in SK-BR-3 cells
(1) Protein printing method
A. The 6-well plate was inoculated with a cell suspension (SK-BR-3), and the plates were pre-incubated in an incubator for 24h (37 ℃ C., 5% CO)2)。
B. Adding 250nM HApt-tFNA, free HApt, and culturing in incubator for 48h (37 deg.C, 5% CO)2)。
C. Extracting the whole protein of all samples, and detecting the change of HER2 protein by Western blot. The brief detection procedure is as follows: pouring glue → loading sample → electrophoresis → transferring membrane → sealing liquid is sealed for 1h → first anti 4 ℃ overnight → recycling first anti → TBST washing 3 times (5-10 min each time) → second antibody incubation 1h → abandoning second antibody, TBST washing 3 times (5-10 min each time) → exposure.
As a result: as shown in FIGS. 12D-E, HApt-tFNA can significantly reduce the amount of HER2 protein in SK-BR-3 cells compared with free HApt, further indicating that the inhibition of the growth of SK-BR-3 cells and the promotion of the apoptosis of SK-BR-3 cells are in certain correlation with the number of HER2 protein in the cells.
(2) Immunofluorescence technique
A. The cell suspension (SK-BR-3) is inoculated into a confocal dish and placed in an incubator for 24 h. Adding 250nM HApt-tFNA, free HApt, and culturing in incubator for 48h (37 deg.C, 5% CO)2). The medium was aspirated off, washed 3 times with PBS, 5min each;
fixing with 4% paraformaldehyde for 25min, removing paraformaldehyde by suction, washing with PBS for 5min for 3 times;
treating with 0.5% Triton-100 for 20-25min, removing Triton-100, washing with PBS for 3 times (5 min each);
D. treating sheep serum for 1h, removing sheep serum by suction, washing with PBS for 3 times, each for 5 min;
E. primary anti (anti-HER2 antibody) treatment was performed at 4 ℃ overnight. The next day, rewarming at 37 ℃ for 0.5h, recovering the primary antibody, washing with PBS 3 times, 5min each time. Treating the secondary antibody carrying fluorescence at 37 deg.C in dark for 1 hr, removing the secondary antibody, washing with PBS for 3 times, each for 5 min;
F. treating phalloidin in dark for 10-30min, removing phalloidin, washing with PBS for 5min for 3 times;
DAPI treatment, protected from light, 10min, aspiration of DAPI, washing 3 times with PBS, 5min each. Sealing with 10% glycerol, and storing at 4 deg.C in dark. And (6) performing detection on the machine.
As a result: as shown in FIG. 12F, HApt-tFNA can significantly reduce the fluorescence intensity of HER2 protein of SK-BR-3 cells compared with free HApt, which further indicates that the inhibition of the growth of SK-BR-3 cells and the promotion of the apoptosis of SK-BR-3 cells have a certain correlation with the number of HER2 protein in the cells.
The experimental example shows that HApt-tFNA can inhibit proliferation of HER2 positive cells and promote apoptosis of HER2 positive cells, and the process is related to HER2 protein (inhibited).
EXAMPLE 4 Studies of lysosomal pathways of action
Comparison of lysosome entry of HApt-tFNA with free HApt, and entry of Cy5-tFNA into lysosome
Immune tracing technology:
A. inoculating cell suspension (SK-BR-3) into the confocal dish, and pre-culturing in incubator for 24h (37 deg.C, 5% CO)2)。
B. Cy5-HApt-tFNA, free Cy5-HApt and Cy5-tFNA were added at a concentration of 250nM and incubated in an incubator for 12 hours (37 ℃, 5% CO)2)。
C. Adding lysosome staining agent into the culture medium, and culturing in incubator for 30min (37 deg.C, 5% CO)2)。
After fixation with D.4% paraformaldehyde for 25min, the paraformaldehyde was aspirated and washed with PBS 3 times for 5min each.
DAPI treatment, protected from light, 10min, aspiration of DAPI, PBS wash 3 times, 5min each. Sealing with 10% glycerol, and storing at 4 deg.C in dark. And (6) performing detection on the machine.
As a result: cy5-HApt-tFNA, free Cy5-HApt all entered the lysosomes of SK-BR-3 cells as shown in FIG. 15A, but Cy5-HApt-tFNA entered significantly more than free Cy 5-HApt; as shown in FIG. 16, Cy5-tFNA could enter lysosomes of SK-BR-3 cells.
Observation of HApt-tFNA into lysosomes at different times
Immune tracing technology:
A. inoculating cell suspension (SK-BR-3) into the confocal dish, and pre-culturing in incubator for 24h (37 deg.C, 5% CO)2)。
B. Adding Cy5-HApt-tFNA with a concentration of 250nM, and culturing in incubator for 6, 12, 24, and 36h (37 deg.C, 5% CO)2)。
C. Adding lysosome staining agent into the culture medium, and culturing in incubator for 30min (37 deg.C, 5% CO)2)。
After fixation with D.4% paraformaldehyde for 25min, the paraformaldehyde was aspirated and washed with PBS 3 times for 5min each.
DAPI treatment, protected from light, 10min, aspiration of DAPI, PBS wash 3 times, 5min each. Sealing with 10% glycerol, and storing at 4 deg.C in dark. And (6) performing detection on the machine.
As a result: as shown in FIG. 15B, Cy5-HApt-tFNA had not entered lysosomes of SK-BR-3 cells after 6h of treatment; after 12h of treatment, Cy5-HApt-tFNA was abundantly incorporated into lysosomes of SK-BR-3 cells; after 24h of treatment, Cy5-HApt-tFNA remained in the lysosomes of SK-BR-3 cells, but the number was reduced, i.e., degraded; after 36h of treatment, only a small amount of Cy5-HApt-tFNA remained. In conclusion, it can be seen that: degradation of Cy5-HApt-tFNA was closely associated with lysosomes.
3. Observe whether Cy5-HApt-tFNA and HER2 have a consensus
Immunofluorescence and tracing techniques:
A. inoculating cell suspension (SK-BR-3) into the confocal dish, and pre-culturing in incubator for 24h (37 deg.C, 5% CO)2)。
B. Adding Cy5-HApt-tFNA with a concentration of 250nM, and culturing in incubator for 12, 24h (37 deg.C, 5% CO)2)。
C. Adding lysosome coloring agent into culture mediumCulturing in incubator for 30min (37 deg.C, 5% CO)2)。
After fixation with D.4% paraformaldehyde for 25min, the paraformaldehyde was aspirated and washed with PBS 3 times for 5min each.
E.0.5% Triton-100 for 20-25min, blotted to remove Triton-100, washed 3 times with PBS, 5min each
F. Treating sheep serum for 1h, removing sheep serum by suction, washing with PBS for 3 times, 5min each time
G. Primary anti (anti-HER2 antibody) treatment was performed at 4 ℃ overnight. The next day, rewarming at 37 ℃ for 0.5h, recovering the primary antibody, washing with PBS 3 times, 5min each time. Treating the secondary antibody carrying fluorescence at 37 deg.C in dark for 1 hr, removing the secondary antibody, washing with PBS for 3 times, each for 5 min;
DAPI treatment, protected from light, 10min, blotted off DAPI, washed 3 times with PBS, 5min each. Sealing with 10% glycerol, and storing at 4 deg.C in dark. And (6) performing detection on the machine.
As a result: as shown in FIG. 15C, Cy5-HApt-tFNA was abundantly incorporated into SK-BR-3 cells after 12h of treatment; after 24h of treatment, Cy5-HApt-tFNA remained partially in SK-BR-3 cells, but the number was reduced, i.e., degraded; the HER2 protein was also present in greater numbers after 12h treatment, but the HER2 number was also significantly reduced after 24h treatment. As shown in FIG. 17, the inventors have seen the composite bubble structure of HER2-Cy5-HApt-tFNA in a slice at the bottom of FIG. 15C.
This example shows that Cy5-HApt-tFNA can carry HER2 into lysosomes.
Experimental example 5 further validation of lysosomal inhibitors "lysosomal pathways of action"
CCK-8 experiment
The set groups are respectively: control group (without any treatment); HApt-tFNA group (HApt-tFNA only treatment for 48 h); lysosomal inhibitor (chloroquinine) + HApt-tFNA group (24 h after chloroquinine treatment followed by 48h with HApt-tFNA treatment). The manner of operation is as described above.
As a result: as shown in fig. 18A, the lysosomal inhibitor (chloroquinine) + HApt-tFNA group showed less significant inhibition of cellular activity than the HApt-tFNA group.
Changes in the HER2 protein
The set groups are respectively: control group (without any treatment); HApt-tFNA group (HApt-tFNA only treatment for 48 h); lysosomal inhibitor (chloroquinine) + HApt-tFNA group (24 h after chloroquinine treatment followed by 48h with HApt-tFNA treatment). Western blotting and immunofluorescence techniques were performed as described above.
As a result: as shown in fig. 18B-C, the change in HER2 protein in the lysosomal inhibitor (chloroquinine) + HApt-tFNA group was less pronounced than in the HApt-tFNA group; as shown in fig. 18D, the fluorescence intensity of HER2 protein was significantly stronger in the lysosomal inhibitor (chloroquinine) + HApt-tFNA group than in the HApt-tFNA group.
Taken together, the HER2 changes were closely associated with lysosomes, i.e. HApt-tFNA mediated degradation of HER2 protein by lysosomes.
Experimental example 6 changes in P13K/AKT Signaling pathway
It has been reported in the literature that the PI3K/AKT pathway is active in HER2 positive cells. The present invention allows reduction of HER2 protein, therefore, the inventors provide this experimental example to verify whether the PI3K/AKT signaling pathway is inhibited.
1. Western blotting method
A. The 6-well plate was inoculated with a cell suspension (SK-BR-3), and the plates were pre-incubated in an incubator for 24h (37 ℃ C., 5% CO)2)。
B. Adding 250nM HApt-tFNA, free HApt, and culturing in incubator for 48h (37 deg.C, 5% CO)2)。
C. Extracting the whole protein of all samples, and detecting the change of HER2 protein by Western blot. The brief detection procedure is as follows: pouring glue → loading sample → electrophoresis → transferring membrane → sealing liquid is sealed for 1h → first anti 4 ℃ overnight → recycling first anti → TBST washing 3 times (5-10 min each time) → second antibody incubation 1h → abandoning second antibody, TBST washing 3 times (5-10 min each time) → exposure.
As a result: as shown in FIGS. 19A-F, the changes of P-AKT, Bax, Bcl-2, etc. protein indicate that PI3K/AKT signal pathway is inhibited, i.e. the apoptosis of SK-BR-3 cells is promoted, and the promotion effect of HApt-tFNA is obviously stronger than that of free HApt; the expression of T-GSK and Cyclin-D1 protein is inhibited, namely the proliferation of SK-BR-3 cells is promoted, and the promoting effect of HApt-tFNA is obviously stronger than that of free HApt.
2. Real-time quantitative fluorescent PCR experiment
A. The 6-well plate was inoculated with a cell suspension (SK-BR-3), and the plates were pre-incubated in an incubator for 24h (37 ℃ C., 5% CO)2)。
B. Adding 250nM HApt-tFNA, free HApt, and culturing in incubator for 48h (37 deg.C, 5% CO)2)。
C. Extracting RNA of each group, obtaining cDNA through a reverse transcription kit, and detecting the expression of genes related to a PI3K/AKT cell pathway by using a dye method to perform fluorescent quantitative PCR (polymerase chain reaction): bax, Bcl-2, Cyclin-D1.
As a result: as shown in FIG. 19G-I, the changes of the genes such as Bax, Bcl-2, Cyclin-D1 are consistent with the changes of the corresponding proteins, further indicating that the I3K/AKT signal pathway is inhibited, the apoptosis of SK-BR-3 cells is promoted, and the promotion effect of HApt-tFNA is obviously stronger than that of free HApt; and the proliferation of SK-BR-3 cells is promoted, and the promotion effect of HApt-tFNA is obviously stronger than that of free HApt.
In conclusion, the HApt-tFNA or Cy5-HApt-tFNA of the invention can specifically inhibit HER2 positive tumor cells, and has good application value in the preparation process of related anti-cancer drugs.
SEQUENCE LISTING
<110> Sichuan university
<120> an anti-tumor nano-drug
<130> GYKH1118-2019P014878CC
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 63
<212> DNA
<213> Artificial sequence
<400> 1
atttatcacc cgccatagta gacgtatcac caggcagttg agacgaacat tcctaagtct 60
gaa 63
<210> 2
<211> 63
<212> DNA
<213> Artificial sequence
<400> 2
acatgcgagg gtccaatacc gacgattaca gcttgctaca cgattcagac ttaggaatgt 60
tcg 63
<210> 3
<211> 63
<212> DNA
<213> alcohol dehydrogenase 1-F(artificial sequence)
<400> 3
actactatgg cgggtgataa aacgtgtagc aagctgtaat cgacgggaag agcatgccca 60
tcc 63
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<211> 63
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<213> Artificial sequence
<400> 4
acggtattgg accctcgcat gactcaactg cctggtgata cgaggatggg catgctcttc 60
ccg 63
<210> 5
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<213> Artificial sequence
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gcagcggtgt gggggcagcg gtgtgggggc agcggtgtgg gg 42
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<213> Artificial sequence
<400> 6
gcagcggtgt gggggcagcg gtgtgggggc agcggtgtgg ggttttacgg tattggaccc 60
tcgcatgact caactgcctg gtgatacgag gatgggcatg ctcttcccg 109
Claims (9)
1. An anti-tumor complex, comprising a DNA tetrahedral structure and HApt in a 1: 1 molar ratio;
the HApt is a single-chain nucleic acid molecule which can target, recognize and bind to HER2 protein and mediate HER2 protein to enter a cell lysosome for degradation;
the HApt is connected to 1 single strand of 4 single strands forming a DNA tetrahedral structure through a covalent bond; the 4 single-chain sequences are different;
the sequence of the HApt is the sequence of SEQ ID NO. 5;
the 4 single-chain sequences are selected from the sequences of SEQ ID NO. 1-4 one to one.
2. The anti-tumor complex of claim 1, wherein the HApt is linked to a single strand that further comprises a linking sequence.
3. The anti-tumor complex of claim 1 or 2, wherein a fluorescent molecule Cy5 is further attached to the 5' end of any single strand of the DNA tetrahedral structure.
4. The method for preparing the anti-tumor composition according to claims 1 to 3, wherein 4 single strands of the DNA tetrahedron are maintained at a temperature sufficient for denaturation for 10min or more, and then the temperature is lowered to 2 to 8 ℃ for 20min or more;
1 of the 4 single strands is linked to a HApt sequence.
5. The method for preparing an anti-tumor composition according to claim 4, wherein the 4 DNA tetrahedrons are maintained at 95 ℃ for 10-15 min, and then the temperature is lowered to 4 ℃ for 20 min.
6. Use of the anti-tumor compound of claims 1-3 in the preparation of an anti-tumor medicament.
7. The use according to claim 6, wherein the antineoplastic drug is a drug against HER2 positive tumors.
8. An antitumor drug characterized by being prepared by using the antitumor compound of any one of claims 1 to 3 as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
9. The anti-neoplastic drug of claim 8, wherein said anti-neoplastic drug is a drug that is resistant to HER2 positive tumors.
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